Minerals
All
microorganisms require certain mineral elements for growth and metabolism. Magnesium, phosphorous, potassium, sulphur,
calcium and chlorine are essential components to be added while media preparation. The concentration of phosphate in a medium
may be larger than the other media components, a part of this phosphate is used
as a buffer to adjust pH variation during the fermentation process. Others such
as cobalt, copper, iron, manganese, molybdenum and zinc are present in
sufficient quantities in the water supplies and as impurities in other media
ingredients. For example, corn steep liquor contains a wide range of minerals
that will usually satisfy the minor and trace mineral needs. Occasionally, levels of calcium, magnesium,
phosphorous, potassium, sulphur and chloride ions are too low to fulfil
requirements and these may be added as specific salts. Trace elements such as
manganese, iron and zinc are reported to be important in secondary metabolite
production.
Chelators
The
insoluble metal phosphates form white precipitates while preparing or autoclaving
many media. This can be avoided by the addition of chelating agents such as EDTA,
citric acid, polyphosphates. They form complexes with metal ions such as iron,
calcium, manganese and zinc. These can then be gradually utilised by microorganism.
It is important to check the concentration of chelators otherwise it may
inhibit the growth. In many media these are added separately after autoclaving.
In large scale fermentations, complex ingredients such as crude industrial byproducts
or yeast extract, peptone etc. complex with these metal ions ensuring their slow
release.
Buffers
The
control of pH is extremely important if optimal productivity is to be achieved.
pH variations occur during fermentation as metabolism proceeds and different
acid/alkaline products are released in the media. This can further interfere
with the growth and product formation. Buffers help to maintain the desired pH.
A compound may be added to the medium to serve as a buffer and it may also be
used as a nutrient source. Many media incorporate calcium carbonate to maintain
the pH at 7.4. Carbonate will decompose if pH decreases. Phosphate added as a
media component play an important role in buffering. However high phosphate concentrations
can be critical in many secondary metabolite fermentations, so should be taken
care of.
Buffering
capacity can be provided by the balanced use of the carbon and nitrogen sources
also such as corn steep liquor, peptone etc. which contains proteins, peptides
and amino acids in addition to any contaminating carbon. pH may also be controlled by the addition of
ammonia or sodium hydroxide and sulphuric acid.
Antifoams
Antifoams
are necessary to reduce foam formation during fermentation. Foaming is largely due to media proteins that
become attached to the air-broth interface where they denature to form a stable
foam “skin” that is not easily disrupted.
The foam may block air filters, resulting in the loss of aseptic
conditions, if uncontrolled. The fermenter can become contaminated and
microorganisms are released into the environment. It is important to provide a headspace/ “freeboard”
in fermenters to provide space for the foam generated.
Foaming
can be minimized by (i) the use of a defined medium and a modification of some
of the physical parameters, e.g. pH, temperature, aeration and agitation (if
the foam is due to media components), (ii) use of mechanical foam breakers (iii)
addition of chemical antifoams.
Antifoams
are surface active agents that reduce the surface tension in the foams and
destabilize the protein films or break the foam.
The
ideal antifoam should have the following properties:
·
readily and rapidly dispersed with rapid
action
·
high activity at low concentrations
·
prolonged action
·
non-toxic to fermentation microorganisms,
humans or animals
·
low cost
·
thermostability
· compatibility with other media components
and the process, i.e., having no effect on oxygen transfer rates or downstream
processing operations
·
be heat sterilisable
Natural
antifoams include plant oils (e.g., from soya, sunflower and rapeseed),
deodorized fish oil, mineral oils and *tallow. The synthetic antifoams are
mostly silicon oils, poly alcohols and alkylated glycols. Since antifoams are of low solubility, they
need a carrier, e.g., *lard oil, liquid paraffin or castor oil, which may be
metabolised. The concentrations of many
antifoams which are necessary to control foaming may reduce the oxygen transfer
rate by as much as 50%. Thus, antifoam
addition should be kept to an absolute minimum.
Some antifoams may reduce the oxygen transfer rate as well as adversely
affect downstream processing steps, especially membrane filtration. If the oxygen transfer rate is too severely
affected mechanical foam breakers may have to be considered.
*Tallow is fat from beef
Oxygen
Oxygen
is very critical in fermentation as it can affect the growth rate and product formation.
Depending on the amount of oxygen required by the organism, it may be supplied
in the form of air containing about 21% (v/v) oxygen or occasionally as pure
oxygen when requirements are particularly high.
The organism’s oxygen requirements may vary widely depending upon the
carbon source. The rate of metabolism of
media components, the viscosity of the medium and the presence of antifoams (surface
active agents and can interfere with oxygen transfer) all can influence the
oxygen availability in a medium. For most fermentations the air or oxygen
supply is filter sterilized prior to being injected into the fermenter.
The
specific oxygen uptake rate of a microorganism increases with increase in the
dissolved oxygen concentration up to a certain point referred to as the critical level (Ccrit). Maximum biomass
production is achieved by maintaining the dissolved oxygen concentration
greater than the critical level thus satisfying the organism’s maximum specific
oxygen demand. However, frequently the
objective of a fermentation is to produce a product and not biomass. So, metabolic
disturbance of the cell by oxygen starvation may enhance product formation.
References
- Industrial Microbiology:
An Introduction. Michael J. Waites, Neil L. Morgan, John S
- Principles of Fermentation Technology- Peter Stanbury,
Allan Whitaker, Stephen Hall
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